Preservation of bioactive materials by freeze dried foam

ABSTRACT

This invention provides methods and compositions to preserve bioactive materials in a dried foam matrix. Methods provide non-boiling foam generation and penetration of preservative agents at temperatures near the phase transition temperature of the membranes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application claiming benefit of andpriority of a prior parent utility application Ser. No. 11/978,948,“Dried Foam Preservation of Bioactive Materials”, filed Oct. 29, 2007,which is a Continuation application claiming priority to and benefit ofa prior to parent Utility application Ser. No. 11/520,503 “Dried FoamPreservation of Bioactive Materials a Divisional Application” by VuTruong-Le, filed Sep. 12, 2006 (now U.S. Pat. No. 7,381,425); which is aDivisional application from U.S. Utility application Ser. No.10/412,630, “Dried Foam Preservation of Bioactive Materials” by VuTruong-Le, filed Apr. 10, 2003 (now U.S. Pat. No. 7,135,180), whichclaims priority to and benefit of prior U.S. Provisional Application No.60/372,236, “Formulations and Methods for Preparation” by Vu Truong-Le,filed Apr. 11, 2002. The full disclosure of the prior application isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention is in the field of preservation of biologicmaterials in storage. In particular, the invention relates to, e.g.,preservation of bioactive molecules and viable membranous biologics byglassification in a protective dry foam matrix.

BACKGROUND OF THE INVENTION

Biological materials, such as proteins, eukaryotic cells, bacteria andviruses, are generally unstable when stored in media or other liquidsolutions. For example, enveloped viruses such as live influenza virusmanufactured from egg allantoid fluid loose one log of potency, definedas Tissue Culture Infectious Dose (TCID50), in less than two to threeweeks when stored under refrigerated temperature, i.e. approximately 4°C. At room temperature conditions (approximately 25° C.) and at warmertemperatures such as 37° C., the virus looses the such potency in amatter of days to hours, respectively. Lyophilization processes, whereaqueous formulas are frozen and then dried by sublimation, are commonlyused to stabilize these biological materials. Removal of water andsubstitution of protectant molecules, such as carbohydrates, canincrease stability by preventing chemical degradation, denaturation, andgrowth of microbial contaminants.

In lyophilization (freeze-drying), the biological material is commonlymixed as a solution or suspension with protective agents, frozen, andthen dehydrated by sublimation and secondary drying. The lowtemperatures of freezing and drying by sublimation can slow the kineticsof degradation reactions. However, the low temperatures and low surfaceto volume ratios involved can require long drying time periods. Oftensignificant structural damage results in conventional freeze dryingprocesses due to the slow freezing rate and the length of time bioactivematerial is kept in a frozen state. This damage can involvedenaturation, aggregation, and other untoward physical stresses stemmingfrom the ice crystal structures that are formed during the icenucleation and propagation steps. For this reason, biomaterials thatpossess a cell wall or lipid membrane pose a significant challenge topreserving the bioactivity of larger and more complex entities such asviruses, bacteria, and cells.

Additionally, even under optimal freeze drying conditions, damage canoccur during the secondary drying step. A recent study has suggestedfreeze drying induced damage occurs primarily during the secondarydehydration step when the last remaining amount of water is removed(Webb, S. D. Effects of annealing lyophilized and spray-lyophilizedformulations of recombinant human interferon-gamma. J Pharm Sci 2003April; 92(4):715-29). Therefore, there is sufficient evidence to showthat lyophilization and secondary drying processes can force a proteinor cell, for example, to undergo significant chemical and physicalchanges. Such changes can result in loss of activity of the protein dueto concentration of salts, precipitation/crystallization, shear stress,pH extremes, and residual moisture remaining through the freeze-drying.

Protective agents are chemicals that are added to a formulation toprotect cells and molecules during freezing and to enhance stabilityduring storage. For example, stabilizers for live virus vaccinesgenerally include high concentrations of sugars such as sucrose,mannitol, or sorbitol to improve virus stability during lyophilizationand storage. However, with membrane viruses, and other membranousbiologicals, the protective agents may not penetrate adequately toprotect active molecules within the membrane volume. Therefore asignificant challenge remains to develop an optimal drying process andformulation to achieve adequate stability for thermally labilebiologics.

Some of the problems with lyophilization are overcome by certain dryfoam preservation processes. In U.S. Pat. No. 5,766,520, Preservation byFoam Formation, to Bronshtein, for example, biological solutions orsuspensions in a solvent are thickened by first drying under a moderatevacuum before application of a strong vacuum to cause frothy boiling ofthe remaining solvent to form a dry stable foam. Normally, such boilingis avoided in processing of biological materials due to the oxidationand denaturation that can occur on bubble surfaces. In addition,boiling, even under vacuum, requires input of heat, which can endangerthe stability of the bioactive material. These problems are reduced inBronshtein by including protective agents, such as carbohydrates andsurfactants, in the solution or suspension. Dry foam preservationprocesses of this type have the advantage of faster drying due toconvection of the liquid during boiling and the large surface areapresented by the foam. Reconstitution of such a dry foam can be rapiddue to the presence of the hydrophilic protective agents and the largefoam surface area. The dry foam can be milled to a fine powder tofurther improve reconstitution times or for administration of thebiological material by inhalation.

The dry foam preservation processes described above is limited in itsflexibility to protect a variety of biological materials. For example,the process rules out a freezing step and subsequent sublimation of theice as a means to remove water from the foam. In the case of highlythermolabile materials, a freezing step can provide stability over thecourse of dehydration. Because freezing is avoided in Bronshtein, theformulation must be thickened before foaming and drying so that largeamounts of water are not lost, along with latent heat, to freeze thefoam. The avoidance of freezing requires the process to be conducted atlower vacuum level (7-24 Torr) than in conventional freeze drying orspray freeze drying process cycles. Boiling in Bronshtein, requiresinput of significant, and possibly destabilizing, amounts of heat toprovide the necessary eruption of foam.

The Bronshtein dry foam process is not particularly well adapted topreservation of biological materials having lipid membranes. Forexample, the process is not well adapted to preservation of membranousbiologicals, such as liposomes, viruses or viable cells. Lipid membranesoften prevent penetration of the protective agents into enclosed volumesor prevent adequate removal of water from the enclosed volume. Withoutadequate penetration of protective agents, enzymatic processes, such asproteolysis, and chemical processes, such as oxidation and free radicalattacks, can destroy the activity or viability of the membranousbiological material. Hypoosmotic fluids remaining within membraneenclosed volumes can promote instability of the biological material.

A need remains for methods to preserve biological materials, such asproteins and membranous materials in storage, particularly attemperatures above freezing. Methods to prepare dry foam preservationmatrices through processes with optional freezing and optional boilingsteps, are desirable to suit the sensitivities of particular biologicmaterials. Compositions that can protect such biologicals in storagewould provide benefits in medicine and scientific research. The presentinvention provides these and other features that will become apparentupon review of the following.

SUMMARY OF THE INVENTION

The present invention includes methods and compositions for preservingbioactive materials in storage. The methods generally provide, e.g.,processes of expanding a formulation of the bioactive material and apolyol into a foam followed by drying the foam into a stable dry foamcomposition. The methods can variously include, e.g., freezing of thefoam before drying, inclusion of foaming agents in the formulation,holding the formulation at the phase transition temperature of a lipidmembrane to enhance penetration of protective agents, and/or expansionof the formulation at pressures between about 200 Torr and 25 mTorr.

A stable dry foam composition to preserve bioactive materials havinglipid membranes can be prepared using the methods of the invention. Themethods generally include, e.g., preparing a formulation of a polyol orpolymer in a solvent (such as water or an alcohol) with the bioactivematerial, cooling the formulation to a temperature of about the phasetransition temperature of the lipid membrane, expanding the formulationinto a foam, and drying the foam by evaporation or sublimation toprepare a stable dry foam composition of the lipid membrane containingbioactive material. For example, a formulation of a live attenuatedinfluenza virus with about 40% sucrose, 5% gelatin, 0.02% blockcopolymers of polyethylene and polypropylene glycol, and 25 mM 7.2 pHKP0₄ buffer can be aliquoted into glass lyophilization vials, cooled ata phase transition temperature of about 15° C. for about 30 minutes,expanded in a vacuum of about 50 mTorr for about one hour, and exposedto a drying temperature of about 33° C. for about 48 hours beforesealing the vials. The dry foam composition prepared by such a processcan remain stable for at least about 2 years in storage at about 25° C.

Another method of the invention calls for expanding the formulation intoa foam at vacuum pressures higher than those described in the prior art.For example, a formulation of a bioactive material (including membranesor not) in a solvent with a polyol or polymer, can be expanded into afoam (without requiring foaming agents) by exposure to a pressure lessthan 25 Torr, less than 8 Torr, less than 400 mTorr, or between about200 mTorr and 25 mTorr, and the foam physically stabilized and dried byevaporation or sublimation of the solvent from the foam to prepare a dryfoam composition.

Foaming agents can be provided in methods and formulations of theinvention to provide adequate foaming under conditions not described inthe prior art. In the prior art, foaming action can be rapid, violent,and difficult to control. Furthermore, the foams described elsewhere arealmost exclusively closed cell, i.e. the roof of the foam structure is acontinuous layer with no openings to provide for transfer of heat andmoisture. This can result in foams lacking uniformity in moisturecontent and glass transition properties. In the present invention, aformulation (suspension or solution) of a bioactive material, a foamingagent, and a polyol or polymer in a solvent can be prepared, theformulation expanded into a foam with the bubbles generated by boilingor provided by the action of a foaming agent, and the foam can bestabilized and dried by evaporating or sublimating the solvent from thefoam. Foaming agents can provide bubbles for expansion of formulations,e.g., generating gas bubbles in situ and/or by providing a suspension ofsmall bubbles for expansion under a vacuum. For example, bubbles can begenerated by boiling the agent, degassing the formulation, acidifying acarbonate, hydrating an active metal, electrolysis of water, and/or thelike. Bubbles can be suspended in the formulation before expansion by,e.g., boiling, degassing, chemical generation of gasses, mechanicalwhipping of the formulation, injection of bubbles into the formulation,and/or the like. For example, a formulation of a bioactive material(including membranes or not) in a solvent with a polyol or polymer, canbe expanded into a foam (by the action of the foaming agents) byexposure to a pressure less than 400 Torr, or between about 200 Torr and25 Torr, or between 25 Torr and 7.7 Torr, and the foam physicallystabilized and dried by evaporation or sublimation of the solvent fromthe foam to prepare a dry foam composition.

Methods of the invention can provide a lyophilized dry foam compositionby freeze drying expanded foams. A formulation of bioactive material, apolyol or a polymer can be prepared, the pressure reduced on theformulation to expand a foam, the foam frozen, and the frozen foam driedby sublimation to provide a lyophilized dry foam composition. In methodswhere formulations are frozen, the freezing can be, e.g., by loss oflatent heat, and/or conduction to a cold solid or fluid environment.

Compositions of the invention can be prepared, e.g., according to themethods of the invention above. Formulations for preparation of thecompositions can include, e.g., a solvent, a bioactive material, apolyol, a polymer, a foaming agent, a surfactant and/or a buffer. Totalsolids (e.g., formulation constituents other than solvents) in theformulations can range, e.g., from less than about 30 weight percent toabout 70 weight percent.

Bioactive materials preserved by the methods of the invention caninclude, e.g., peptides, proteins, nucleic acids, antibodies, vaccines,bacteria, viruses, liposomes, platelets, cell suspensions and/or thelike. The viruses can be, e.g., live viruses, attenuated viruses, and/ornon-viable viruses, such as influenza virus, parainfluenza virus, AAV,adenovirus, respiratory syncytial virus, SARS (severe acute respiratorysyndrome) virus, herpes simplex virus, cytomegalovirus, corona virusfamily members, human metapneumovirus, Epstein-Barr virus, and/or thelike. Viruses in formulations of the methods can be present, e.g., in anamount ranging about 10³ TCID₅₀/mL to about 10¹² TCID₅₀/mL, or fromabout 10⁶ TCID₅₀/mL to about 10⁹ TCID₅₀/mL. Dried foam compositions ofthe invention can provide virus present in an amount, e.g., from about10¹ TCID₅₀/g to not more than 10¹² TCID₅₀/g. Dried foam compositions canprovide virus present in an amount, e.g., of about 10² TCID₅₀/g, about10² TCID₅₀/g, about 10³ TCID₅₀/g, about 10⁴ TCID₅₀/g, about 10⁵TCID₅₀/g, about 10⁶ TCID₅₀/g, about 10⁷ TCID₅₀/g, about 10⁸ TCID₅₀/g,about 10⁹ TCID₅₀/g, about 10¹⁰ TCID₅₀/g, or about 10¹¹ TCID₅₀/g.

The polyols of the invention can include, e.g., non-reducing sugars,reducing sugars, sugar alcohols and sugar acids. Polyols can act as,e.g., protective agents and structural constituents of stabilized foamin the invention. Polyols can include, e.g., sucrose, trehalose,sorbose, melezitose, sorbitol, stachyose, raffinose, fructose, mannose,maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose andglucose, mannitol, xylitol, erythritol, threitol, sorbitol, glycerol,L-gluconate, and/or the like. The polyol can be present in theformulation in an amount ranging from about 1 weight percent to about 45weight percent, about 5 weight percent to about 40 weight percent, or atabout 20 weight percent.

Polymers can be present in the formulations and compositions of theinvention to provide, e.g., stability to bioactive materials and to actas structural constituents in the dried foam compositions. Polymers ofthe methods and compositions can include, e.g., hydrolyzed gelatin,unhydrolyzed gelatin, collagen, chondroitin sulfate, a sialatedpolysaccharide, actin, myosin, water soluble polymers such as polyvinylpyrrolidone, microtubules, dynein, kinetin, human serum albumin, and/orthe like. The polymers can be present, e.g., in the formulations of themethods in an amount ranging from about 1 weight percent to about 10weight percent. In one embodiment the polymer is human serum albuminpresent in the formulation at about 5 weight percent.

Surfactants can be present in the formulations and compositions of theinvention, e.g., to stabilize and enhance the solubility of otherconstituents. Surfactants of the formulations and compositions caninclude, e.g., polyethylene glycol, polypropylene glycol, polyethyleneglycol/polypropylene glycol block copolymers, polyethylene glycol alkylethers, polypropylene glycol alkyl ethers, polyethyleneglycol/polypropylene glycol ether block copolymers, alkylarylsulfonates,phenylsulfonates, alkyl sulfates, alkyl sulfonates, alkyl ethersulfates, alkyl aryl ether sulfates, alkyl polyglycol ether phosphates,polyaryl phenyl ether phosphates, alkylsulfosuccinates, olefinsulfonates, paraffin sulfonates, petroleum sulfonates, taurides,sarcosides, fatty acids, alkylnaphthalenesulfonic acids,naphthalenesulfonic acids, lignosulfonic acids, condensates ofsulfonated naphthalenes with formaldehyde, or condensates of sulfonatednaphthalenes with formaldehyde and phenol, lignin-sulfite waste liquor,alkyl phosphates, quaternary ammonium compounds, amine oxides, betaines,and/or the like. Tween® and Pleuronic® surfactants, such as, e.g.,polyethylene glycol sorbitan monolaurate, polyoxyethylenesorbitanmonooleate, or block copolymers of polyethylene and polypropyleneglycol, are particularly preferred surfactants of the invention.Surfactants can be present in formulations of the invention in amountsof about 0.01 weight percent to about 1 weight percent.

Buffers can be included in formulations and compositions of theinvention, e.g., to stabilize other constituents, control pH, and/or toparticipate in foaming processes. Typical buffers of the invention are,e.g., potassium phosphate, sodium phosphate, sodium acetate, sodiumcitrate, sodium succinate, histidine, imidazole, ammonium bicarbonate,or a carbonate. pH levels can be adjusted in the formulations,compositions, and reconstituted products of the invention, e.g., to a pHranging from about pH 4 to about pH 10, from about pH 6 to about pH 8,and, more typically, near neutral or about pH 7.2.

In methods of the invention, formulations can be cooled, e.g., beforeexpansion, to thicken the formulation, to effect freezing duringpreliminary drying, to enhance penetration of protective agents throughlipid membranes at phase transition temperatures, and/or the like.Cooling to the phase transition temperature of lipid membranes caninvolve, e.g., adjusting the formulation to a temperature ranging fromabout 2° C. to about 70° C., 10° C. to 45° C., or about 12° C. to about16° C. To obtain adequate penetration of protective agents, such aspolyols and/or polymers, the formulation can be held at the phasetransition temperature for about 10 minutes to about 60 minutes, or forabout 30 minutes.

In methods of the invention, formulations can be held, e.g., intemperature controlled and/or pressure controlled chambers. Duringexpansion, foam stabilization, primary drying, and/or secondary dryingstages, the pressure of gasses in the environment of the formulationscan be reduced to less than about 400 Torr, about 200 Torr or less,between about 100 Torr and about 25 Torr or less, between 25 Torr and7.7 Torr or less, between 2500 mTorr and about 50 mTorr, or about 25mTorr or less. The vacuum can be maintained, e.g., for expansion, foamstabilization, or primary drying for a time ranging from about one hourto about two hours.

Secondary drying of the dry foam can proceed, e.g., to further reduceresidual moisture in the stabilized foam and/or dry foam of the methodsand compositions of the invention. For example, secondary drying can beinitiated by increasing the temperature of the formulation to a dryingtemperature that is less than or about the glass transition temperatureof the dry foam. Drying temperatures can range, e.g., from about 10° C.to about 70° C., or from about 30° C. to about 35° C. Moisture can beremoved from the gaseous environment around the foam, e.g., bydesiccation and/or condensation, to help drive the drying process to adesired end point. In secondary drying, the dried foam can be held,e.g., at reduced pressure and at the drying temperature for a timeranging from about 6 hours to about 5 days, or about 48 hours. Secondarydrying can continue, e.g., until the residual moisture content of thestable composition ranges from about 0.1% to about 5%.

To provide convenient and stable dosage forms, formulations can befilled into suitable containers. Containers can be provided with etchedbottoms, e.g., promote bubble formation at the bottom of the containerand/or to generate an open cell foam during foam expansion processsteps. The container can be aseptically sealed, e.g., with a stopper toretain a vacuum and/or inert gas environment over the stablecompositions of the invention.

In one embodiment, the composition of the invention can be a dry foamcomposition of a bioactive material comprising a lipid membrane enclosedcompartment and a polyol or polymer that has penetrated the lipidmembrane into the enclosed compartment. In this embodiment, the polyoland/or polymer protective agents penetrated the lipid membrane while atabout the phase transition temperature of the membrane.

The compositions of the invention can be prepared, e.g., by the methodsof the invention. For example, a dry foam composition with improvedstability and shelf-life can be prepared by: preparing a formulation ofa bioactive material (with or without lipid membranes) a polyol or apolymer, with or without a foaming agent; optionally, cooling theformulation to a temperature of about a phase transition temperature ofany relevant lipid membranes; reducing pressure (optionally, from about200 Torr to about 25 Torr, 7.7 Torr, 2.5 Torr, 50 mTorr, or less) on theformulation to form a foam, which is optionally frozen and the icesublimated; drying the foam to stabilize the physical structure and/orto provide a dry foam composition.

Compositions of the invention can be administered to mammals, such ashumans, as vaccines, therapeutics, pharmaceuticals, and/or the like. Thecompositions can be administered, e.g., as ground powders by inhalationor as reconstituted liquids by injection. The dry foam compositions ofthe invention can be ground, e.g., to a stable powder compositions withany desired size range, for example, an average particle size from about0.1 um to about 150 um, or about 10 um to about 100 um, for quickreconstitution or delivery by inhalation. Reconstituted liquid can beadministered by, e.g., delivering the composition to the mammal byintravenous, intramuscular, intraperitoneal, intracerebrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, intranasal, and/or pulmonary routes. Administering bioactivematerial in methods and compositions of the invention typicallyinvolves, e.g., delivery of a dose ranging from about 0.01 ng/kg toabout 15 mg/kg.

Definitions

It is to be understood that this invention is not limited to particulardevices or biological systems, which can, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a”, “an” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “asurface” includes a combination of two or more surfaces; reference to“bacteria” includes mixtures of bacteria, and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used inaccordance with the definitions set out below.

“Ambient” temperatures or conditions are those at any given time in agiven environment. Typically, ambient room temperature is 22° C.,ambient atmospheric pressure, and ambient humidity are readily measuredand will vary depending on the time of year, weather conditions,altitude, etc.

“Boiling” refers, e.g., to the rapid phase transition from liquid to gasthat takes place when the temperature of a liquid is above its boilingtemperature. The boiling temperature, as is well known to those skilledin the art, is the temperature at which the vapor pressure of a liquidis equal to the applied pressure.

“Buffer” refers to a buffered solution that resists changes in pH by theaction of its acid-base conjugate components. The pH of the buffer willgenerally be chosen to stabilize the active material of choice, and willbe ascertainable by those in the art. Generally, this will be in therange of physiological pH, although some proteins, can be stable at awider range of pHs, for example acidic pH. Thus, preferred pH ranges arefrom about 1 to about 10, with from about 3 to about 8 beingparticularly preferred; more preferably, from about 6.0 to about 8.0;yet more preferably, from about 7.0 to about 7.4; and most preferably,at about 7.0 to about 7.2. Suitable buffers include a pH 7.2 phosphatebuffer and a pH 7.0 citrate buffer. As will be appreciated by those inthe art, there are a large number of suitable buffers that may be used.Suitable buffers include, but are not limited to, potassium phosphate,sodium phosphate, sodium acetate, histidine, imidazole, sodium citrate,sodium succinate, ammonium bicarbonate and carbonate. Generally, buffersare used at molarities from about 1 mM to about 2 M, with from about 2mM to about 1 M being preferred, and from about 10 mM to about 0.5 Mbeing especially preferred, and 25 to 50 mM being particularlypreferred.

“Degassing” refers to the release of a gas from solution in a liquidwhen the partial pressure of the gas is greater than the appliedpressure. If water is exposed to nitrogen gas at one atmosphere (about760 Torr), and the partial pressure of nitrogen in the waterequilibrates to the gas phase pressure, nitrogen can bubble from thewater if the gas pressure is reduced. This is not boiling, and can oftenoccur at pressures above a pressure that would boil a solvent. Forexample, bottled carbonated soft drinks, with a high partial pressure ofCO₂ gas, bubble rapidly (but do not boil) when pressure is reduced byremoving the bottle cap.

“Dispersibility” means the degree to which a powder composition can bedispersed (i.e. suspended) in a current of air so that the dispersedparticles can be respired or inhaled into the lungs of a subject. Thus,a powder that is only 20% dispersible means that only 20% of the mass ofparticles can be suspended for inhalation into the lungs.

“Dry” in the context of dried foam compositions refers to residualmoisture content less than about 10%. Dried foam compositions arecommonly dried to residual moistures of 5% or less, or between about 3%and 0.1%. A “dry foam” can be a stabilized foam with less than 10%residual moisture content, a foam after primary drying, and/or a foamafter secondary drying. “Dry” in the context of particles for inhalationmeans that the composition has a moisture content such that theparticles are readily dispersible in an inhalation device to form anaerosol.

“Excipients” or “protectants” (including cryoprotectants andlyoprotectants) generally refer to compounds or materials that are addedto ensure or increase the stability of the therapeutic agent during thespray freeze dry process and afterwards, for long term stability andflowability of the powder product. Suitable excipients are generallyrelatively free flowing particulate solids, do not thicken or polymerizeupon contact with water, are basically innocuous when inhaled by apatient and do not significantly interact with the therapeutic agent ina manner that alters its biological activity. Suitable excipients aredescribed below and include, but are not limited to, proteins such ashuman and bovine serum albumin, gelatin, immunoglobulins, carbohydratesincluding monosaccharides (galactose, D-mannose, sorbose, etc.),disaccharides (lactose, trehalose, sucrose, etc.), cyclodextrins, andpolysaccharides (raffinose, maltodextrins, dextrans, etc.); an aminoacid such as monosodium glutamate, glycine, alanine, arginine orhistidine, as well as hydrophobic amino acids (tryptophan, tyrosine,leucine, phenylalanine, etc.); a methylamine such as betaine; anexcipient salt such as magnesium sulfate; a polyol such as trihydric orhigher sugar alcohols, e.g. glycerin, erythritol, glycerol, arabitol,xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol;Pluronics; surfactants; and combinations thereof.

“Glass” or “glassy state” or “glassy matrix,” refers to a liquid thathas lost its ability to flow, i.e. it is a liquid with a very highviscosity, wherein the viscosity ranges from 10¹⁰ to 10¹⁴pascal-seconds. It can be viewed as a metastable amorphous system inwhich the molecules have vibrational motion but have very slow (almostimmeasurable) rotational and translational components. As a metastablesystem, it is stable for long periods of time when stored well below theglass transition temperature. Because glasses are not in a state ofthermodynamic equilibrium, glasses stored at temperatures at or near theglass transition temperature relax to equilibrium and lose their highviscosity. The resultant rubbery or syrupy, flowing liquid is oftenchemically and structurally destabilized. While a glass can be obtainedby many different routes, it appears to be physically and structurallythe same material by whatever route it was taken. The process used toobtain a glassy matrix for the purposes of this invention is generally asolvent sublimation and/or evaporation technique.

The “glass transition temperature” is represented by the symbol T_(g)and is the temperature at which a composition changes from a glassy orvitreous state to a syrup or rubbery state. Generally T_(g) isdetermined using differential scanning calorimetry (DSC) and isstandardly taken as the temperature at which onset of the change of heatcapacity (Cp) of the composition occurs upon scanning through thetransition. The definition of T_(g) is always arbitrary and there is nopresent international convention. The T_(g) can be defined as the onset,midpoint or endpoint of the transition; for purposes of this inventionwe will use the onset of the changes in Cp when using DSC and DER. Seethe article entitled “Formation of Glasses from Liquids and Biopolymers”by C. A. Angell: Science, 267, 1924-1935 (Mar. 31, 1995) and the articleentitled “Differential Scanning calorimetry Analysis of GlassTransitions” by Jan P. Wolanczyk: Cryo-Letters, 10, 73-76 (1989). Fordetailed mathematical treatment see “Nature of the Glass Transition andthe Glassy State” by Gibbs and DiMarzio: Journal of Chemical Physics,28, NO. 3, 373-383 (March, 1958). These articles are incorporated hereinby reference.

“Penetration enhancers” are surface active compounds that promotepenetration of a drug through a mucosal membrane or lining and can begenerally used where this feature is desirable, e.g., intranasally,intrarectally, and intravaginally.

“Pharmaceutically acceptable” excipients (vehicles, additives) are thosewhich can reasonably be administered to a subject mammal to provide aneffective dose of the active ingredient employed. Preferably, these areexcipients which the Federal Drug Administration (FDA) have to datedesignated as ‘Generally Regarded as Safe’ (GRAS).

“Pharmaceutical composition” refers to preparations which are in such aform as to permit the biological activity of the active ingredients tobe unequivocally effective, and which contain no additional componentswhich are toxic to the subjects to which the composition would beadministered.

A “polyol” is a substance with multiple hydroxyl groups, and includes,e.g., sugars (reducing and nonreducing sugars), sugar alcohols and sugaracids. Preferred polyols herein have a molecular weight which is lessthan about 600 kDa (e.g. in the range from about 120 to about 400 kDa).A “reducing sugar” is a polyol which contains a hemiacetal group thatcan reduce metal ions or react covalently with lysine and other aminogroups in proteins. A “nonreducing sugar” is a sugar which does not havethese properties of a reducing sugar. Examples of reducing sugars arefructose, mannose, maltose, lactose, arabinose, xylose, ribose,rhamnose, galactose and glucose. Nonreducing sugars include sucrose,trehalose, sorbose, melezitose and raffinose. Mannitol, xylitol,erythritol, threitol, sorbitol and glycerol are examples of sugaralcohols. As to sugar acids, these include L-gluconate and metallicsalts thereof.

A “Powder” is a composition that consists of finely dispersed solidparticles that are relatively free flowing and capable of being readilydispersed in an inhalation device and subsequently inhaled by a patientso that the particles are suitable for intranasal or pulmonaryadministration via the upper respiratory tract including the nasalmucosa.

“Recommended storage temperature” for a composition is the temperature(T_(b)) at which powdered drug composition can be stored to maintain thestability of the drug product over the shelf life of the composition inorder to ensure a consistently delivered dose. This temperature isinitially determined by the manufacturer of the composition and approvedby the governmental agency responsible for approval the composition formarketing (e.g., the Food and Drug Administration in the U.S.). Thistemperature will vary for each approved drug product depending on thetemperature sensitivity of the active drug and other materials in theproduct. The recommended storage temperature will vary from about 0° toabout 40° C., but generally will be ambient temperature, i.e. about 25°C. Usually a drug product will be kept at a temperature that is at orbelow the recommended storage temperature.

A biologically active material “retains its biological activity” in apharmaceutical composition, if the biological activity of thebiologically active material, such as an enzyme, at a given time iswithin about 10% (within the errors of the assay) of the biologicalactivity exhibited at the time the pharmaceutical composition wasprepared as determined in a binding assay, for example. In the case ofliving viruses or bacteria, biological activity can be consideredretained when the viral titer or colony count of the composition iswithin one log of the initial titer or count. For live cells, thebiological activity is considered retained when the live cell count ofthe composition is within 50% of the initial count. The assay that isused to determine live influenza virus titer is the Fluorescent FocusAssay (FFA assay). The titer from this assay is reported as logFluorescent Focus Unit per milliliter (log FFU/ml). One log FFU/ml isapproximately equal to one log Tissue Culture Infectious Dose per ml(log TCID50/ml). Other “biological activity” assays are elaboratedbelow.

A biologically active material “retains its chemical stability” in apharmaceutical composition, if the chemical stability at a given time issuch that the biologically active material is considered to retain itsbiological activity as defined herein. Chemical stability can beassessed by detecting and quantifying chemically altered forms of thebiologically active material. Chemical alteration may involve sizemodification (e.g. clipping of proteins) which can be evaluated usingsize exclusion chromatography, SDS-PAGE and/or matrix-assisted laserdesorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS),for example. Other types of chemical alteration include chargealteration (e.g. occurring as a result of deamidation) which can beevaluated by ion-exchange chromatography, for example.

A biologically active material “retains its physical stability” in apharmaceutical composition if it shows no significant increases inaggregation, precipitation and/or denaturation upon visual examinationof color and/or clarity, or as measured by UV light scattering or bysize exclusion chromatography.

A “stable” formulation or composition is one in which the biologicallyactive material therein essentially retains its physical stabilityand/or chemical stability and/or biological activity upon storage.Various analytical techniques for measuring stability are available inthe art and are reviewed, e.g., in Peptide and Protein Drug Delivery,247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs.(1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stabilitycan be measured at a selected temperature for a selected time period.Trend analysis can be used to estimate an expected shelf life before amaterial has actually been in storage for that time period. For liveinfluenza viruses, stability is defined as the time it takes to loose 1log of FFU/ml or 1 log of TCID50/ml. Preferably, the composition isstable at room temperature (˜25° C.) for at least three months, or at40° C. for at least 1 month, and/or stable at about 2-8° C. for at least1 year. Furthermore, the composition is preferably stable followingfreezing (to, e.g., −70° C.) and thawing of the composition.

In a pharmacological sense, a “therapeutically effective amount” of abiologically active material refers to an amount effective in theprevention or treatment of a disorder wherein a “disorder” is anycondition that would benefit from treatment with the biologically activematerial. This includes chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

“Unit dosage” refers to a receptacle containing a therapeuticallyeffective amount of a composition of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart of temperature and pressure versus time during anexemplary foam drying process.

FIGS. 2A to 2D are photographic images of a formulation foam drying inglass vials.

FIG. 3 shows the phase transitions of FluMist™ A/Sydney Influenza virusvaccine using Fourier transformed infrared spectroscopy (FTIR).

FIG. 4 shows process activity (viral titer) loss for B/Harbin influenzavirus vaccine after the freeze dry foaming process where primary dryingwas conducted at 10° C., 15° C., and 20° C.

FIG. 5 shows the stability trend of B/Harbin influenza virus vaccinewhich was subjected to the freeze dry foaming process where primarydrying was conducted at 10° C., 15° C., or 20° C.

FIG. 6 shows the stability trend for potency of foam dried B/Ann Arborinfluenza virus in storage at 37° C. and 50° C.

FIG. 7 shows the stability trends for three different strains ofinfluenza virus vaccine foam dried in formulation AVS53.

FIG. 8 shows X-ray diffraction data and an electron micrographdemonstrating the amorphous nature of a foam composition of theinvention.

DETAILED DESCRIPTION

The methods and compositions of the present invention can provideextended storage of bioactive materials, such as, e.g., peptides,proteins, nucleic acids, antibodies, vaccines, bacteria, viruses,liposomes, platelets, and/or cell suspensions, in a glassy matrix of adry foam. Methods of the invention provide a dry foam preservativecomposition by, e.g., preparing a formulation comprising a bioactivematerial with a polyol or polymer in a solvent (with or without afoaming agent), reducing pressure to expand the formulation into a foam(by degassing, boiling, and/or expansion of introduced bubbles), andstabilizing the foam by evaporating or sublimating the solvent from thefoam (with or without freezing the foam). In methods, e.g., particularlywell suited to preservation of bioactive materials comprising lipidmembranes, the bioactive material can be formulated into a suspensionwith protective agents, precooled, and held at a temperature near thephase transition temperature of the membrane to allow the protectiveagents to penetrate the membrane before expanding the formulation into afoam.

Methods of Preparing Stable Dry Foams

Methods for preparing stable dry foams for preservation of bioactivematerials includes, in general, e.g., preparation of a formulationcombining the bioactive material with a polyol and/or polymer in asolution or suspension, reducing the pressure applied to the formulationto initiate foaming, stabilizing the foam by removal of a solvent, anddrying the foam.

In one embodiment, for example, a formulation of bioactive material,polyol and/or polymer, in a solvent, is expanded to a foam under apressure of between about 200 Ton and about 25 Torr before stabilizingand drying the foam. This embodiment is distinguished from prior artdiscussed above, e.g., in not requiring a strong vacuum (pressure 24Torr or less) in order to obtain adequate foam expansion. In thisembodiment, adequate foaming can be obtained at higher pressures becausethe methods of the invention provide foam expansion, e.g., fromdegassing of saturated gasses from the formulation, boiling of highvapor pressure solvents from the formulation, gas forming chemistries,and/or enlargement of bubbles injected or trapped in the formulation.Formulations of this embodiment can be, e.g., precooled and/or losesubstantial latent heat during expansion of foam or drying to, e.g.,optionally result in freezing and/or lyophilization of the foam. Afterthe primary drying stage is complete, the stabilized dry foam can beheld, e.g., at secondary drying temperatures at pressures below 50mTorr, to complete drying of the formulation.

In another embodiment, a foaming agent is present in the formulation,e.g., to provide foam expansion and/or control with or without boiling.For example, a formulation containing a foaming agent, a bioactivematerial, and a polyol and/or polymer, can be subjected to a reducedpressure in which the formulation is expanded into a foam (by action ofthe foaming agent), stabilized and dried. The foaming agent can be,e.g., gas in solution in the formulation, a high vapor pressure(volatile) solvent, a carbonate, an active metal, a direct electriccurrent, a suspension of fine gas bubbles, and/or the like, as describedbelow in the Foaming the Formulation section.

Another embodiment of the invention provides, e.g., methods to preparelyophilized foam compositions for preservation of bioactive materials.For example, a formulation containing a bioactive material, and a polyoland/or polymer, can be expanded into a foam under reduced pressure,frozen and sublimated to provide a lyophilized dry foam composition.Freezing, in this embodiment, can be, e.g., by conduction of heat awayfrom the formulation, and/or by loss of latent heat due to solventevaporation or sublimation.

The present invention includes, e.g., methods for preservation ofbiological materials having a lipid membrane component. Methods forpreserving bioactive materials comprising lipid membranes can include,e.g., cooling a membranous biologic material formulation to a membranephase transition temperature of about 45° C. to 0° C. for about 30minutes in a solution containing about 2% to 40% of a polyol protectiveagent. (The protective agents can, e.g., penetrate the membranes inphase transition to stabilize biological molecules within enclosedvolumes. A membrane phase transition temperature is the temperature atwhich the lipid membrane transitions between a fluid (high mobility)phase to a more rigid gel-crystalline phase. It is postulated thatbecause lipid membranes tend to be pervious to passive diffusion ofexternal milieu at lipid bilayer's characteristic phase transitiontemperature, one way to load stabilizers/protective agents to the cells,bacteria, or viruses is by preincubating at such phase transition.)Pressure can then be reduced, e.g., to boil the formulation and producea foam. Water can be rapidly lost from the formulation, along withlatent heat, resulting, e.g., in freezing of the foam. Water continuesto be lost, e.g., by sublimation over the course of several minutes toprovide a substantially dry foam composition. The temperature can bewarmed, e.g., to drive off additional residual moisture and water ofhydration to enhance the physical and/or chemical stability of the dryfoam.

FIG. 1 shows an exemplary freeze foam drying process of the invention.Superimposed on a chart of vacuum chamber temperatures and pressuresversus time are photographic images of formulations containing amembrane virus at various stages of drying. Chamber temperature line 11indicates the temperature of the vacuum chamber during the freezefoaming process. The chamber temperature is held at about the phasetransition temperature of the virus, or about 15° C., throughpenetration stage 12, foaming stage 13, and initial drying stage 14. Thechamber temperature is ramped up to drying temperatures of about 33° C.during secondary drying phase 15. Chamber pressure line 16 remains at orabove atmospheric pressure during the penetration stage, drops to about2500 mTorr during the foaming stage, about 250 mTorr during the initialdrying stage, and about 50 mTorr during the secondary drying stage. Vialtemperature lines 17 represent temperatures measured from thermocouplesplaced into formulations in representative vials during the process. Thevials hold the membrane phase transition temperature during thepenetration stage but chill suddenly as the pressure drops during thefoaming stage due to loss of latent heat from evaporation andsublimation of water from the formulation. Vial temperatures graduallyrise to near chamber drying temperatures as the rate of residualmoisture loss tapers off in the secondary drying stage.

FIGS. 2A to 2D show photographic images of representative vials offormulation during stages of freeze foam drying. In FIG. 2A, liquidformulation at the bottom of the vial is beginning to boil as pressurein the chamber begins to drop. In FIG. 2B, a foamy matrix has begun tostabilize as it thickens with loss of water and lower temperatures. InFIG. 2C, the foam is frozen and has lost most of the initial dryingstage water. FIG. 2D shows the dried foam glassy matrix well into thesecondary drying stage.

In one embodiment of the method, for example, the formulation includes alive attenuated influenza virus bioactive material in a solution of 40%sucrose, 5% gelatin, 0.02% Pluronic F68, and a pH 7.2 phosphate buffer.The formulation is aliquoted into sterile 10 ml siliconized glass vialsand precooled to 15° C. (about the phase transition temperature of thevirus membrane, see FIG. 3) for about 30 minutes. The pressure israpidly reduced to about 50 mTorr for about an half an hour to generatethe foam with ice nucleation and ice propagation throughout. After theinitial foaming and freezing, ice sublimation and evaporation produce aphysically stable foam. (such a foam can be generated at vacuums betweenabout 400 Torr and 7.7 Torr or less, or 2.5 Torr to about 50 mTorr, whenthe formulation contains foaming agents). The temperature is increasedto about 33° C. for about 2 days in a secondary drying step to reducethe residual moisture of the composition to a desired level. The vialsare aseptically sealed to keep out contaminants and moisture forstability in storage.

Preparing a Formulation

Formulations of the invention can include, e.g., a bioactive materialformulated into a solution or suspension containing a polyol, polymer,foaming agent, surfactant, and/or a buffer. The formulation ingredientscan be combined in a sequence using techniques appropriate to theconstituents, as is appreciated by those skilled in the art. Forexample, the polymers and/or high concentrations of polyols can bedissolved into a heated aqueous solution with agitation before coolingand admixture with the bioactive material. The bioactive material, suchas a virus or bacterium, can be, e.g., concentrated and separated fromgrowth media by centrifugation or filtration before resuspension intothe formulation.

The bioactive material can be, e.g., a material of interest thatprovides any bioactivity, such as, e.g., enzymatic activity, storage ofgenetic information, an affinity interaction, induction of immuneresponses, cellular multiplication, infection, inhibition of cellgrowth, stimulation of cell growth, therapeutic effects, pharmacologiceffects, antimicrobial effects, and/or the like. For example, thebioactive materials can be, enzymes, antibodies, hormones, nucleicacids, bacteria, viruses, liposomes, platelets, other cells, and/or thelike. The bioactive material can be, e.g., living cells and/or viableviruses. The bioactive material can be, e.g., nonliving cells orliposomes useful as vaccines or delivery vehicles for therapeuticagents. Viral bioactive materials of the invention can be, e.g., liveviruses such as, influenza virus, parainfluenza virus, AAV, adenovirus,respiratory syncytial virus, herpes simplex virus, cytomegalovirus, SARSvirus, corona virus family members, human metapneumovirus, Epstein-Banvirus, and/or the like.

The protective agents of the methods can include, e.g., any of a varietyof polyols. For example, the polyol, such as sucrose, can physicallysurround the bioactive material to promote retention of molecularstructure throughout the drying process and impart structural rigidityto the glassy matrix in the dry state. Other functions of the polyol caninclude, e.g., protecting the bioactive material from exposure todamaging light, oxygen, moisture, and/or the like For example, thepolyol, such as sucrose, can physically surround and protect thebioactive material from exposure to damaging light, oxygen, moisture,and/or the like. The polyols can, e.g., replace water of hydration lostduring drying, to prevent denaturation of biomolecules of the material.In the methods of the invention, polyols can provide, e.g., a thickenerwith tenacity to foster formation and stabilization of bubbles that formthe dry foam structure of the preservative compositions. Although theinvention is not limited to any particular polyols, the formulation andfoam compositions can include, e.g., sucrose, trehalose, sorbose,melezitose, sorbitol, stachyose, raffinose, fructose, mannose, maltose,lactose, arabinose, xylose, ribose, rhamnose, galactose, glucose,mannitol, xylitol, erythritol, threitol, sorbitol, glycerol,L-gluconate, and/or the like. Most polyols can be readily dissolved formixture into the formulation in amounts ranging, e.g., from about 1weight percent to about 45 weight percent, about 2 weight percent toabout 40 weight percent, or about 5 weight percent to about 20 weightpercent.

Polymers can be included in the formulations of the method, e.g., toprovide protective benefits. As with polyols, polymers can provide,e.g., physical and chemical protection to the bioactive materials. Thepolymers can often provide, e.g., more thickening viscosity by weight tothe formulation than simple polyols. The linear or branching strands ofpolymers can provide, e.g., increased structural strength to the driedfoam compositions of the invention. Many polymers are, e.g., highlysoluble in water, so they do not significantly hinder reconstitution ofdry foams. Polymer protective agents, in the methods of the inventioncan include, e.g., hydrolyzed gelatin, unhydrolyzed gelatin, watersoluble polymers such as polyvinyl pyrrolidone, ovalbumin, collagen,chondroitin sulfate, a sialated polysaccharide, actin, myosin,microtubules, dynein, kinetin, human serum albumin, and/or the like.

Foaming agents can be, e.g., formulation constituents capable of causingexpansion of the formulation into a foam on application of reducedpressure. Foaming agents can be, e.g., small bubbles suspended in theformulation which can expand on application of reduced pressure and/orconstituents capable of generating gas bubbles in the formulation.Foaming agents can be, e.g., gasses in solution, gas forming chemicals,readily boiling solvents, entrapped or suspended bubbles, injectedbubbles, and/or the like.

Surfactants can be included in the formulations of the methods toprovide, e.g., increased solubility to other formulation constituents,protection against surface tension induced denaturation of certainbiomolecules during foaming, bubble stabilization, fasterreconstitution, and/or the like. The surfactants can be, e.g., suitableionic or non-ionic detergents, Tween surfactants, Pluronic surfactants,and/or the like.

Buffers can be added to the formulations of the method, e.g., to providea suitable stable pH to the formulations of the method and compositionsof the invention. Typical buffers of the invention include, e.g.,potassium phosphate, sodium phosphate, sodium acetate, histidine,imidazole, sodium citrate, sodium succinate, ammonium bicarbonate,and/or a carbonate. The buffers can be adjusted to the appropriate acidand salt forms to provide, e.g., pH stability in the range from about pH4 to about pH 10. A pH near neutral, such as, e.g., pH 7.2, is preferredfor many compositions.

Other excipients can be included in the formulation. For example, aminoacids, such as arginine and methionine can be constituents of theformulation and compositions. The amino acids can, e.g., act aszwitterions that block charged groups on processing surfaces and storagecontainers preventing nonspecific binding of bioactive materials. Theamino acids can increase the stability of compositions by, e.g.,scavenging oxidation agents, scavenging deamidation agents, andstabilizing the conformations of proteins. In another example, glycerolcan be included in the formulations of the invention, e.g., to act as apolyol and/or plasticizer in the dried foam composition. EDTA can beincluded in the composition, e.g., to scavenge metal ions that caninitiate destructive free radical chemistries.

Cooling the Formulation

Formulations of the invention can be cooled before foam expansion, foamstabilization, freezing, and/or drying, to provide benefits, such as,e.g., stabilization of bioactivity, thickening of the formulation,enhanced penetration of formulation constituents through membranes,and/or freezing the formulation before lyophilization.

Cooling can be by any appropriate technique known in the art. Forexample, cooling can be by contact and conduction with refrigeratedhardware, contact with streams of cold fluids, loss of latent heat,and/or the like. Typically, formulations are held in glass containers onracks within a temperature controlled process chamber where theyequilibrate to the controlled temperature. The chamber can include,e.g., pressure control capabilities so that cooling can be driven byloss of latent heat from evaporation or sublimation of formulationsolvents.

The formulations of the invention can be, e.g., precooled to the phasetransition temperature of biological material associated lipid membranesto enhance the penetration of protective agents. The lipid bilayers ofbiological membranes, and monolayers of some liposomes, can exist in afluid phase at temperatures above the main phase transition temperature(T_(m)) and as a crystalline phase at temperatures below the T_(m).Fluid phase membranes and crystalline phase membranes can present acontinuous hydrophobic barrier to penetration by hydrophilic molecules.Without being bound to any particular theory, it is believed that attemperatures near the T_(m), transmembrane defects can exist at theboundaries between regions of fluid and crystalline phases on a lipidmembrane. Such transmembrane defects can provide increased permeabilityto hydrophilic molecules, such as many protective agents of theinvention. Moreover, because the formulation has a high solids content,a chemical gradient is produced which further drives the solutes, suchas protective agents, into the membrane. When moisture is later removedfrom the formulation, the protective agents can be retained within themembrane enclosed volume at stabilizing levels. Enhanced processstability and storage stability for virus exposed to protective agentsat the membrane phase transition temperature (see, FIG. 3) are shown inFIGS. 4 and 5, respectively.

The T_(m) of many lipid membranes is above the freezing temperature andthe glass transition temperatures (T_(g)) of formulations of theinvention. This allows ready diffusion of protective agents in liquidsolution through lipid membranes at about their T_(m). For example, a40% solution of sucrose remains liquid for effective penetration of acell at a typical membrane T_(m) of 15° C. Increased permeability ofprotective agents in liquid solution through membranes in phasetransition can protect biologic molecules within membrane enclosedvolumes.

The T_(m) of a membrane can be determined, e.g., Fourier TransformedInfrared (FTIR) microscopy, bending rigidity methods, ionic permeabilitystudies, and the like. In methods of the invention, formulations ofbioactive materials with lipid membranes can be held, e.g., attemperatures between about 0° C. to about 70° C., about 2° C. to about45° C., about 12° C. to 16° C., or about 15° C. Live influenza virusfoam dried at the putative phase transition temperature of 15° C. (see,FIG. 3) was found to be more resistant to process-related potency loss(see, FIG. 4) and exhibited significantly better long term stability atroom temperature than that foam dried at 10° C. and 20° C. (see, FIG.5). Formulations can be held at the T_(m) to allow adequate penetrationof protective agents. For example formulations can be held at the lipidmembrane T_(m) for about 10 to about 60 minutes, or about 30 minutes.

Foaming the Formulation

Foaming can result, e.g., from entrapment of gases released from theformulation and/or expansion of preexisting bubbles in suspension. Forexample, as gas pressure over the formulation is reduced, the boilingpoint of solvent constituents can drop below the temperature of theformulation, resulting in rapid evaporation or boiling of the solvent.Significant bubbling can also occur in the formulation, e.g., when thepressure is reduced below the partial pressure of gasses dissolved inthe solvent, resulting in bubbles from degassing. Bubble formation canbe chemically induced. Alternatively, bubbling can be physically inducedby introducing a gas through the bottom of the vessel, e.g., such asthrough fritted glass.

Expanding the formulation into a foam can be by, e.g., expansion ofbubbles within the formulation by reduction of applied pressure. Thebubbles can be, e.g., preexisting, injected, and/or generated in situ.The bubbles can be, e.g., suspended within the formulation beforeexpansion, injected into the formulation before or during expansion, orgenerated by boiling, degassing, or gas forming chemical reactions.Formulation constituents, e.g., included to promote expansion of theformulation into a foam can be foaming agents of the invention.

Expansion of the formulation can result, e.g., from boiling of theformulation. Boiling occurs, e.g., at the boiling point of a solvent, orwhen the vapor pressure of a formulation solvent exceeds the surroundingpressure. Boiling can be controlled, e.g., by adjusting the temperatureof the formulation (higher temperatures result in higher vaporpressures) and/or by adjusting the applied pressure. Typically,formulations of the invention can be boiled under reduced pressure(vacuum, or pressure less than atmospheric) to provide a lowered boilingtemperature more conducive to the stability of bioactive materials.Formulations comprising a solvent with a low boiling point (or highvapor pressure) can boil at lower temperatures. For example, inclusionof certain alcohols, ethers, fluorocarbons, and/or the like, can providelowered boiling points for formulations of the invention.

Degassing can, e.g., provide expansion of the formulation into a foam.Gases can diffuse and dissolve into liquid solvents until an equilibriumis established between the partial pressures of gases in the liquid andthe surrounding atmosphere. If the pressure of the surroundingatmosphere is, e.g., suddenly dropped, the gasses can rapidly escape theliquid as bubbles. For example, when an aqueous solution, which has beenequilibrated with a gas at atmospheric pressure, is exposed to a loweredpressure, gas bubbles can form on the walls of the container or canerupt from the solution as a “fizz”. This is not boiling, but is therelease of dissolved gasses from the solution, or degassing. If thepressure is lowered further, the gasses can be substantially removedfrom the solution. Eventually, depending on the solvent, temperature andpressure, the solvent of the solution can begin to boil. Formulations ofthe invention can be expanded into foams under reduced pressure bydegassing. Formulations can be exposed to gasses at suitable pressures,such as about one atmosphere (about 760 Torr at sea level) to about 500atmospheres, to drive gasses into the formulation. Where the gas hasequilibrated with the formulation at high pressures (greater than 1atmosphere) the reduced pressure providing expansion does not have to bea vacuum (less than 1 atmosphere). Where the gas has equilibrated withthe formulation at ambient pressures, or less, the reduced pressureinitiating expansion of bubbles can be, e.g., a vacuum. Gasses that canact as foaming agents of the invention can be any known in the art, suchas, air, nitric oxide, nitrogen, oxygen, low molecular weighthydrocarbons, inert gases, and/or the like.

Chemical reactions, e.g., which generate a gas can provide expansion ofthe formulation into a foam. Foaming agents of the invention can be,e.g., chemicals involved in gas generating chemical reactions, as willbe appreciated by those skilled in the art. For example, a carbonate inthe formulation can react with an acid to produce CO₂ gas. In otherreactions, e.g., active metals, such as sodium or lithium, in thepresence of water can react to provide hydrogen gas. Electrolyticreactions using direct electric currents can be used, e.g., to providehydrogen and/or oxygen gasses at electrodes. Gasses generated within theformulation can, e.g., expand adiabatically or under constant pressureto expand the formulation into a foam. Optionally, gasses chemicallygenerated within the formulation can be expanded by reduction of theapplied pressure to expand the formulation into a foam.

Bubbles can be incorporated in to formulations, e.g., through mechanicalprocesses. Formulations can be expanded into foam, e.g., by forcefulincorporation of gas bubbles into the formulation and/or by expansion ofinjected small bubbles in a reduced pressure. For example, bubbles canbe stirred, whipped, blown, jetted, agitated, sonicated, vortexed,blended, and/or the like, into the formulation. After introduction,e.g., into viscous formulations of the invention the bubbles can remainsuspended for extended periods of time. Suspended bubbles can be, e.g.,expanded by application of reduced pressure and/or stabilized by dryingor cooling of the formulation. In one embodiment, small bubbles (e.g.,0.1 to 1 mm diameter) can be introduced into a formulation by the forceof a pressurized gas through a filter membrane.

Foaming can be initiated or expanded by reducing pressure over theformulation. Foaming can be the result, e.g., from degassing of gassesfrom the formulation, expansion of small incorporated bubbles and/orboiling of the solvent. The escaping gasses can be trapped, e.g., by theviscous protective agents and/or surfactants of the formulation. Thefoaming step can result in, e.g., initial drying of the formulation,thickening and structural stabilization, and/or freezing of the foam.

Processes for preservation of bioactive materials comprising lipidmembranes includes, e.g., a combination of pre-cooling to a phasetransition temperature and vacuum conditions that can result in freezingof the formulation. Because freezing can be a major cause of protein(and membrane damage) during freeze-drying, the prior art teaches theuse of higher pressures (e.g., ˜100 Torr or more), concentratedsolutions, and/or higher initial temperatures to prevent freezing. Theuse of formulations containing various cryoprotective agents and processparameters of the invention can cryoprotect bioactive proteins andmembranous should freezing result during the foam expansion, foamstabilization, or drying stages of the process.

Evaporation of solvent from the formulation can provide acceleratedinitial drying of the formulation under vacuum. The boiling of solventspeeds initial drying of the formulation, e.g., by rapid transfer ofsolvent out of the formulation, convective turn over of the formulation,and by increasing the surface area.

As evaporation proceeds, the foam structure can be stabilized. Assolvent is driven from the formulation, the protective agents insolution can become concentrated and thick. Evaporation of solvent andloss of latent heat can cool the formulation. At some point, the cooledand concentrated protective agents can reach their glass transitiontemperature and stop flowing as a liquid. Loss of latent heat can resultin freezing of the formulation. The glassy and/or frozen formulation canpreserve a stable foam structure. An open cell foam structure can beprovided throughout, e.g., by providing an etched glass bottom to theholding container to promote bubble formation at the bottom of thecontainer. Bubbles traveling up through the thickening formulation canform interconnected spaces of an open cell foam. Open cell foam can alsobe promoted by rapid drying and thickening that prevents settling ofbubble free formulation or formation of a sealing skin over theformulation. Open cell foam can shorten secondary drying times andreconstitution times.

Foaming can be affected by conditions, such as, e.g., types andconcentrations of formulation constituents, formulation temperatures,applied pressure levels, the rate of pressure changes, and/or the like.For example, the presence of surfactants or thickening agents canstabilize bubbles for a less dense foam. In another example, replacementof lost latent heat, e.g., by heating the process chamber, can prolongthe boiling of solvent. In another example, lower pressures can providemore vigorous or continuous boiling. Pressure can be reduced, e.g., toless than about 400 Torr, about 200 Torr or less, between about 100 Torrand about 25 Torr or less, between 25 Torr and 7.7 Torr or less, orbetween 2500 mTorr and about 50 mTorr, or about 25 mTorr or less, toproduce desired foaming and/or freezing in the methods of the invention.The vacuum can be maintained for about 1 hour or about 2 hours, e.g., tocomplete foaming, foam stabilization, and initial drying of the foam.

Initial (primary) drying in the methods of the invention can include,e.g., lyophilization. When latent heat is lost without replacement,e.g., the freezing temperature of the formulation can be reached. Asadditional latent heat is lost due to evaporation and/or sublimation,the formulation can freeze, e.g., stabilizing a foam structure. Initialdrying can continue, e.g., as additional solvent is removed bysublimation into the vacuum. The sublimation and/or evaporation can bedriven, e.g., by removal of solvent (moisture) from the gaseousenvironment around the foam by condensation or desiccation.

Secondary Drying

Secondary drying of the structurally stabilized and initially dried foamcan, e.g., remove entrapped solvent, or water of molecular hydration, toprovide a composition that is stable in storage, e.g., for extendedperiods at ambient temperatures. Secondary drying can involve, e.g.,application of warm temperatures in a strong vacuum for several hours todays.

For example, heat can be added to the initially dried foam to drive offresidual solvent. Heat can be applied, e.g., through heating the reducedpressure atmosphere and/or heat can be conducted to the foam throughassociated hardware, such as shelves, trays, and glass vials. The dryingtemperature can be, e.g., less than the glass transition temperature ofthe remaining composition in order to prevent collapse of the foamstructure. The methods of the invention result in apharmaceutically-acceptable, glassy matrix comprising at least onebiologically active material within the amorphous glassy matrix.Preferably, the composition is almost completely dry. Some water orother aqueous solvent can remain in the composition but typically, notmore than 10% residual moisture remains by weight. The dryingtemperature can range from about 10° C. to about 70° C., about 25° C. toabout 45° C., or about 35° C. A typical secondary drying process caninclude, e.g., raising the temperature to a drying temperature of fromabout 30° C. to about 35° C., and holding for from about 0.5 days toabout 5 days to provide a stable dried foam composition with 0.1% toabout 10%, or about 3% residual moisture. As used herein, “dry”,“dried”, and “substantially dried” encompass those compositions withfrom about 0% to about 5% water. Preferably, the glassy matrix will havea moisture content from about 0.1% to about 3% as measured using theKarl Fisher method.

A vacuum can be provided in the secondary drying process to drive therate of water removal and/or to push removal to lower residual moisturelevels. The vacuum during secondary drying can be, e.g., less than 400Torr, less than 100 Torr, less than 2.5 Ton, less than 500 mTorr, lessthan 100 mTorr, less than 50 mTorr, or preferably about 25 mTorr.

The resulting product from this process is generally an amorphous solid(see, FIG. 8), wherein the glassy excipient material, e.g. sucrose, isin an amorphous glassy state and encases the biologically activematerial, thereby preventing protein unfolding and significantly slowingmolecular interactions or cross-reactivity, due to greatly reducedmobility of the compound and other molecules within the glassycomposition. This process has been postulated to occur either viamechanical immobilization of the protein by the amorphous glass or viahydrogen bonding to polar and charged groups on the protein, i.e. viawater replacement, thereby preventing drying induced denaturation andinhibiting further degradative interactions. As long as the glassy solidis at a temperature below its glass transition temperature and theresidual moisture remaining in the excipients is relatively low, thelabile proteins and/or bioactive material containing lipid membranes canremain relatively stable. It should be noted that achieving a glassystate is not necessarily a prerequisite for long term stability as someactive ingredients may fare better in a more crystalline state. While itis generally recognized that biomaterials are generally easier tostabilize when dried to an amorphous glassy state, there are cases wherethe such glassy state is neither necessary nor sufficient for long termpreservation. It is important to note that the mechanisms attributed tostabilization of biologicals can be multifactorial and not limited tothe amorphous nature of the powder matrix in which the active ingredientis encased. Stabilization under the process described here can involve anumber of factors including but not limited to the reduction inconformational mobility and flexibility of the protein side chainsand/or reduction in the free volume as a result of the encasement,improvement in the structural rigidity of the matrix, reduction in thephase separation of excipient from the active ingredient, improvement inthe degree of water displacement by selecting the optimal hydrogenbonding donor. The latter is a function of the affinity and avidity ofthe excipient for the surface of the protein, nucleic acids,carbohydrate, or lipids being stabilized. In general, as long as thesolid is at a temperature below its glass transition temperature and theresidual moisture remaining in the excipients is relatively low, thelabile proteins and/or bioactive material containing lipid membranes canremain relatively stable.

Filling and Administration

Formulations can be, e.g., filled into containers before foaming anddrying, or aliquoted into individual containers for use, as desired.Formulations can be filled, e.g., into standard glass lyophilizationvials for processing into stabilized foams. The glass vials can besterile with an etched bottom and a hermetically sealable stopper.Bioactive materials of the invention can be administered, e.g., byinjection of reconstituted solutions or suspensions, or inhalation ofground foam powder particles.

The compositions described herein can be stable, i.e., they preserve thebiological activity of the encased biologically active material and arechemically and/or physically stable. The compositions were tested forstability by subjecting them to aging at elevated temperature (e.g., 37°C.) and measuring the biological activity, chemical and/or physicalstability of the formulations. As an example for live attenuatedinfluenza virus vaccine (FluMist™), results of these studies demonstratethat the virus formulated in these formulations were stable for at leastone month at 50° C. and for more than three months at 37° C. (see, FIG.6). Stability is defined as time for one log fluorescent focus unit/ml(FFU/ml) potency loss. At 25° C., the live influenza viruses werestabile for more than one year (see, FIG. 7). Such formulations arestable even when high concentrations of the biologically active materialare used. Thus, these formulations are advantageous in that they may beshipped and stored at temperatures at or above room temperature for longperiods of time.

The stable composition of the bioactive material in an amorphous glassymatrix (see, FIG. 8) provided after drying can be further processedusing methods known in the art. For example, the glass matrix is easilydivisible by cutting, milling, or other dividing techniques. Processesfor grinding or pulverizing drugs are well known in the art. Forexample, a hammer mill, an impact mill known as Entoleter mill, a jetmill, a pin mill, a Wiley mill, or similar milling device can be used.The preferred particle size is less than about 100 um to about 0.1 um,and preferably less than 50 um. Particles less than about 10 um in sizeare suitable, e.g., for pulmonary administration by inhalation, whilelarger particles can be suitable for administration to the upperrespiratory tract and nasal regions. The particle size can be chosen soas to obtain varying dispersion and flowability characteristics. Forexample, free flowing powders may be especially desirable for intranasalor pulmonary delivery. The powdered compositions of the invention can beeasily rehydrated with water, saline, or other fluids.

Dry foam compositions can be reconstituted with a suitable aqueousbuffer for administration by injection or inhalation. For example,compositions of the invention can be administered to a mammal bydelivering the bioactive material through the intravenous,intramuscular, intraperitoneal, intracerebrospinal, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, intranasal,or pulmonary routes. The large surface area of foam and the highsolubility of many protective agents allows dry foams of the inventionto be reconstituted at lower or higher concentrations than the originalformulation. In some cases, e.g., the bioactive material can bereconstituted at high concentrations, such as up to about 400 mg/ml, fordelivery of an adequate dose in a small volume subcutaneous injection.Less concentrated reconstituted solutions can be, e.g., administered asan aerosol by inhalation. The choice of administration route can dependon, e.g., the site of action, pharmacological considerations, and thelike. A typical dose of a bioactive material in the methods of theinvention is from about 0.01 ng/kg to about 15 mg/kg.

The appropriate dosage (“therapeutically effective amount”) of thebiologically active material will depend, for example, on the conditionto be treated, the severity and course of the condition, whether thebiologically active material is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the biologically active material, the type ofbiologically active material used, and the discretion of the attendingphysician. The biologically active material can be suitably administeredto the patent at one time, or over a series of treatments, and may beadministered to the patent at any time from diagnosis onwards. Thebiologically active material may be administered as the sole treatmentor in conjunction with other drugs or therapies useful in treating thecondition in question.

As a general proposition, the therapeutically effective amount of thebiologically active material administered will be in the range of about0.01 ng/kg to about 50 mg/kg of patent body weight whether by one ormore administrations, with the typical range of protein used being about0.05 ng/kg to about 20 mg/kg, more preferably about 0.1 ng/kg to about15 mg/kg, administered daily, for example. However, other dosageregimens may be useful. The progress of this therapy can be monitored byconventional techniques.

In another embodiment of the invention, an article of manufacture isprovided comprising a container which holds the pharmaceuticalcompositions of the present invention and optionally providesinstructions for its use. Suitable containers include, for example,bottles, vials and syringes. The container may be formed from a varietyof materials such as glass or plastic. An exemplary container is a 3-20cc single use glass vial. Alternatively, for a multidose formulation,the container may be 3-100 cc glass vial. The container holds theformulation and the label on, or associated with, the container mayindicate directions for use. The article of manufacture may furtherinclude other materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for use.

Compositions of the Invention

Compositions of the invention include dry foam compositions of abioactive material and a polyol and/or a polymer. Compositions of theinvention can be prepared, e.g., by the methods of the invention.Compositions of the invention can be prepared by, e.g., preparing aformulation of a polyol and a bioactive material with lipid membranes,cooling the formulation to a temperature of about a phase transitiontemperature of the lipid membranes, reducing pressure on the formulationto form a foam, freezing the foam, and sublimating water from the frozenfoam to provide a lyophilized dry foam composition. Secondary dryingconditions can be employed to further dry the foam. Compositions of theinvention include, e.g., reconstituted dry foam in an aqueous buffer.

In one embodiment, the composition of the invention is a vaccine of liveattenuated influenza virus. The composition is prepared, e.g., accordingto the methods of the invention including secondary drying the foam at adrying temperature below the glass transition, e.g., between 30° C. and35° C., for between 6 hours and 5 days, to provide a final compositionwith less than about 5% residual moisture. Such a composition can remainstable in storage at about 25° C. for 1 year or more.

FIGS. 5 and 6 show charts of stability data for an envelope viruspreserved in the dry foam of the invention and stored at 25° C., 37° C.,and 50° C. The charts of potency (fluorescent focus units per mL—FFU/mL)versus time with individual data time-points as dots or squares. Trendlines 51 and 95% confidence intervals 52 indicate a predicted stability(not more than 1 log potency loss) of about 40 months for virus storedin the composition of the invention at 25° C.

In another embodiment, the compositions of the invention include, e.g.,blood platelets preserved in stable storage as bioactive material in adry foam. Blood platelets are cytoplasmic fragments from megakaryocytesthat can aggregate at the site of blood vessel lesions to preventbleeding and initiate repairs. Platelet infusions into patients are usedto correct deficiencies or dysfunctions of circulating platelets as aresult of trauma, disease, or drug induced dysfunction. For example,patients suffering from idiopathic thrombocytopenia, sickle cell anemia,and those undergoing ablative chemotherapy can be treated with plateletinfusions. The increasing use of ablative chemotherapy for a widevariety of malignancies has resulted in an increased need forreplacement platelet therapy. A major difficulty in using isolatedplatelets is their short shelf-life. Platelets are only approved by theFood and Drug Administration (FDA) for storage in a liquid state for upto five days at room temperature, during which time the functionalproperties rapidly deteriorate. This causes many logistic problems inboth civilian and military medicine.

Compositions for preservation of platelets can be prepared by collectingblood into a suitable anticoagulant followed by obtaining platelet richplasma (PRP) by any method known in the art. The platelets can beprocessed according to the methods of the invention to yield a stabledried foam comprising the platelets. Because platelets are large andmore complex structure than a virus, drying at its characteristic phasetransition is particularly important. The dried platelets can bereconstituted by resuspension in a physiologically acceptable bufferbefore infusion. For therapeutic use, the buffer is sterile. The buffercan be any buffer of suitable pH. Preferably, the reconstitution buffercan contain a substance or substances that exhibit high colloidalosmotic pressure, including, but not limited to, polyethylene glycol(PEG) and hydroxy-ethyl starch (HES). Preferably, the buffer is 1-5%human serum albumin (HSA) in saline.

Formulations for Preparation of Dry Foam Compositions

Formulations for preparation of dry foam compositions of the inventioncan include, e.g., bioactive materials, polymers, polyols, foamingagents, surfactants, and/or buffers. Such formulations can be processedaccording to methods of the invention to provide stable compositions forstorage and administration of the bioactive materials.

Bioactive materials of the invention include, e.g., materials withdetectable bioactivity in living systems, biological cells and moleculesused in analysis, biological cells and molecules used in medicine,biological cells and molecules used in research, and/or the like. Forexample, bioactive materials of the compositions of the inventioninclude peptides, proteins, hormones, nucleic acids, antibodies,vaccines, bacteria, viruses, liposomes, platelets, cell suspensions,and/or the like.

Bioactive materials comprising lipid membranes in the compositions aregenerally live, biologically active, viable or non-living, cells,viruses, and/or liposomes. For example the bioactive agents can includevaccines, viruses, liposomes, bacteria, platelets, and cells. Viralbioactive agents can include, e.g., influenza virus, parainfluenzavirus, AAV, adenovirus, respiratory syncytial virus, herpes simplexvirus, SARS virus, human metapnuemovirus, corona virus family members,cytomegalovirus, and/or Epstein-Ban virus which can be present in theformulations of the invention in amounts ranging from about 10¹TCID₅₀/mL or more, from about 10³ TCID₅₀/mL up to about 10¹² TCID₅₀/mL,or from about 10⁶ TCID₅₀/mL to about 10⁹ TCID₅₀/mL. The bioactivematerial will generally be present in an amount of less than about 1%;more preferably, less than about 0.001%; and most preferably, less thanabout 0.0001% by weight.

The formulations for preparation of dry foam compositions can include,e.g., substantial total solids (constituents minus the solvent, such aswater). A major portion of the total solids can comprise the bioactivematerial, a polyol, and/or a polymer. For example, the polyol can bepresent in the formulation in a concentration ranging from about 2weight percent to about 50 weight percent, from about 5 weight percentto about 45 weight percent, or from about 20 weight percent to about 40weight percent. In another example, the polymer can be present in theformulation in a concentration ranging from about 1 weight percent toabout 10 weight percent, or about 5 weight percent. Preferably, theformulation should have a high solids content, typically between about5% and 70%, or between about 30% to 50%. The viscosity of formulationsof the invention are typically greater than 5 centipoise (cP); morepreferably, greater than 10 cP. A preferred formulation exhibits ˜12 cP.

Polyols of the invention can include, e.g., various sugars,carbohydrates, and alcohols. For example, the polyols can includenon-reducing sugars, sucrose, trehalose, sorbose, melezitose, and/orraffinose. The polyols can include, e.g., mannose, maltose, lactose,arabinose, xylose, ribose, rhamnose, galactose and glucose, mannitol,xylitol, erythritol, threitol, sorbitol, glycerol, or L-gluconate. Whereit is desired that the formulation be freeze-thaw stable, the polyol ispreferably one which does not crystallize at freezing temperatures (e.g.−20° C.) such that it destabilizes the biologically active material inthe formulation.

Polymers of the invention can include, e.g., various carbohydrates,polypeptides, linear and branched chain hydrophilic molecules. Forexample, polymers of the formulation can include gelatin, hydrolyzedgelatin, ovalbumin, polyvinylpyrrolidone, collagen, chondroitin sulfate,a sialated polysaccharide, actin, myosin, microtubules, dynein, kinetin,or human serum albumin. These additives do not necessarily solelystabilize the biologically active material against inactivation; theyalso may help to prevent the physical collapse of the freeze-driedmaterial during lyophilization and subsequent storage in the solidstate. Other gelatin substitutes that may also function as stabilizersinclude native collagen and alginate.

Preferably, gelatin and more preferably, hydrolyzed gelatin, is used.“Hydrolyzed gelatin” refers to gelatin that has been subjected topartial hydrolysis to yield a partially hydrolyzed gelatin having amolecular weight of from about 1 kDa to about 50 kDa, or about 3 kDa.This gelatin hydrolysis product has approximately the same amino acidcomposition as gelatin. The typical amino acid composition of hydrolyzedgelatin is known. Partially hydrolyzed gelatin may be obtained from anynumber of commercial sources. Partially hydrolyzed gelatin may also beobtained by enzymatic hydrolysis of gelatin by means of a proteolyticenzyme, such as, for example, papain, chymopapain, and bromelin,although other known hydrolysis means may be employed, e.g., acidhydrolysis. Preferably, a gelatin having a molecular weight of betweenabout 1 kDa and 50 kDa is used. Gelatin hydrolyzed to about 3 kDa orless can be less allergenic than full length gelatin. The gelatin may bederived from a variety of sources, including pig and bovine. Humanizedcollagen as well as highly processed collagen, for example, FreAlagin, apharmaceutical gelatin with reduced allergenicity, available from MiyagiChemical Industrial Co, Ltd., can be used. Again, the amount of gelatinused in the formulation will vary depending on the overall compositionof the formulation and its intended use. Generally, the concentration ofgelatin will be from about 1 to about 7%; more preferably, between about1 and 5%. A preferred formulation comprises about 5% gelatin.

Formulations for preparation of the compositions of the invention caninclude, e.g., one or more surfactants to aid in solubility andstability of formulation constituents. The surfactants can include,e.g., nonionic detergents, such as polyethylene glycol sorbitanmonolaurate (Tween 20), polyoxyethylenesorbitan monooleate (Tween 80),block copolymers of polyethylene and polypropylene glycol (Pluronic),and/or the like. The formulations can include ionic detergents.Formulations and compositions of the invention can include surfactants,such as, e.g., polyethylene glycol, polypropylene glycol, polyethyleneglycol/polypropylene glycol block copolymers, polyethylene glycol alkylethers, polypropylene glycol alkyl ethers, polyethyleneglycol/polypropylene glycol ether block copolymers, alkylarylsulfonates,phenylsulfonates, alkyl sulfates, alkyl sulfonates, alkyl ethersulfates, alkyl aryl ether sulfates, alkyl polyglycol ether phosphates,polyaryl phenyl ether phosphates, alkylsulfosuccinates, olefinsulfonates, paraffin sulfonates, petroleum sulfonates, taurides,sarcosides, fatty acids, alkylnaphthalenesulfonic acids,naphthalenesulfonic acids, lignosulfonic acids, condensates ofsulfonated naphthalenes with formaldehyde, or condensates of sulfonatednaphthalenes with formaldehyde and phenol, lignin-sulfite waste liquor,alkyl phosphates, quaternary ammonium compounds, amine oxides, betaines,and/or the like. Surfactants can be present in formulations of theinvention in a concentration ranging from about 0.001 weight percent toabout 2 weight percent, or about 0.01 weight percent to about 1 weightpercent.

Buffers can be present, e.g., to control pH, enhance stability, affectconstituent solubility, provide comfort on administration, and the like,in formulations for preparation of dry foam compositions. Formulation pHcan be controlled in the range of about pH 4 to about pH 10, from aboutpH 6 to about pH 8, or about pH 7.2. Preferred buffers are often pairedacid and salt forms of a buffer anion generally recognized as safe forthe particular route of administration of the bioactive material.Typical buffers for use in the formulations and compositions of theinvention include, e.g., potassium phosphate, sodium phosphate, sodiumacetate, sodium citrate, histidine, imidazole, sodium, succinate,ammonium bicarbonate, carbonates, and the like.

In one embodiment, the formulation contains the above-identified agents(i.e., biologically active material, polyol, surfactant, and gelatin)and is essentially free of one or more preservatives, such as benzylalcohol, phenoly, m-cresol, chlorobutanol, and benethonium chloride). Inanother embodiment, a preservative may be included in the formulation,particularly when the formulation is a multidose formulation.

One or more pharmaceutically acceptable carriers, excipients, orstabilizers such as those described in Remington's PharmaceuticalSciences 16^(th) Edition, Osol, A. Ed. (1980) may be included in theformulation provided that they do not adversely affect the desiredcharacteristics of the formulation. Acceptable carriers, excipients orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed and include; additional buffering agents; co-solvents;salt-forming counterions such as potassium and sodium; antioxidants,such as methionine, N-acteyl cysteine, or ascorbic acid; chelatingagents, such as EDTA or EGTA. Amino acids, such as, e.g., arginine andmethionine can be included in the formulations. Arginine can be presentin the formulations in an amount ranging from about 0.1 weight percentto about 5 weight percent. Methionine can be present in the formulationin a concentration ranging from about 1 mM to about 50 mM or about 10mM. Glycerol can be present in the formulation in a concentrationranging, e.g., from about 0.1 weight percent to about 5 weight percent,or about 1 weight percent. EDTA can be present in the formulation in aconcentration ranging, e.g., from about 1 mM to about 10 mM, or about 5mM.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Preservation of Live Attenuated Virus

This example describes a composition that maintained protein integrityand stability after storage at 37° C. for 125 days.

Monovalent live attenuated influenza virusB/Harbin (CAZ039 lot) wasformulated as an 8.0 log FFU/ml titer solution (˜10 microgram/ml totalprotein concentration of viral stock solution) containing 40% sucrose,5% gelatin, 0.02% Pluronic F68, 25 mM 7.2 pH KP0₄ buffer. One mLaliquots of this solution were then dispensed into 10 mL glasslyophilization vials, partially covered with lyophilization stoppers,and lyophilized using a VirTis Genesis 25EL lyophilizer (available fromVirTis, Gardiner, N.Y.) according to the following cycle conditions:

1) Pre-cool shelves to 15° C. (with desiccant on lyophilizer shelf withthe condenser set at −60° C.);

2) Load vials and allow to equilibrate for 30 minutes;

3) Set vacuum to 50 mTorr;

4) Hold for 60 minutes;

5) Ramp to 33° C. at about 0.7° C./minute;

6) Hold for 48 hours; and

7) Stopper vials.

The resultant foam had a moisture content of approximately 2.2% (w/w)and a T_(g) of about 43° C.

Example 2 Formulations

The following formulations were prepared according to the methods ofthis invention using B/Harbin influenza virus or placebo. The pH offormulations were adjusted with either sodium hydroxide or potassiumhydroxide.

Polymer ID Glutamate Polyol Additive Surfactant Other AVS1 20% 10% 5%Gelatin 0.1% Pluronic 2% arginine, 5 mM sucrose F68 EDTA, 10 mMmethionine, 50 mM 7.2 KPO4 buffer AVS2 20% 10% — 0.1% Pluronic 5%arginine, 5 mM sucrose F68 EDTA, 10 mM methionine, 7.2 KPO4 buffer 50mM, AVS3 25% 15% — 0.1% Pluronic 5% arginine, 5 mM sucrose F68 EDTA, 50mM 7.2 KPO4 buffer AVS4 25% 15% — 0.1% Pluronic 5% arginine, 50 mMsucrose F68 7.2 KPO4 buffer AVS5 25%  5% — 0.1% Pluronic 5% arginine, 50mM sucrose F68 7.2 KPO4 buffer AVS1A 20 10% 5% Gelatin 0.1% Pluronic 2%arginine, 5 mM sucrose F68 EDTA, 10 mM methionine, 50 mM 7.2 KPO4 bufferAVS2A 20 10% 0.1% Pluronic 5% arginine, 5 mM sucrose F68 EDTA, 10 mMmethionine, 50 mM 7.2 KPO4 buffer AVS3A 25 15% 0.1% Pluronic 5%arginine, 5 mM sucrose F68 EDTA, 10 mM methionine, 50 mM 7.2 KPO4 bufferAVS4A 25 15% 0.1% Pluronic 5% arginine, 50 mM sucrose F68 7.2 KPO4buffer AVS5A 25  5% 0.1% Pluronic 5% arginine, 50 mM sucrose F68 7.2KPO4 buffer AVS6 20 10%  5% Gelatin 0.1% Pluronic 2% arginine, 5 mMsucrose F68 EDTA, 10 mM methionine AVS7 20 10% 0.1% Pluronic 5%arginine, 5 mM sucrose F68 EDTA, 10 mM methionine, 50 mM 7.2 KPO4 bufferAVS8 25 15% 0.1% Pluronic 5% arginine, 5 mM sucrose F68 EDTA, 10 mMmethionine, 50 mM 7.2 KPO4 buffer AVS9 25 15% 0.1% Pluronic 5% arginine,50 mM sucrose F68 7.2 KPO4 buffer AVS10 25  5% 0.1% Pluronic 5%arginine, 50 mM sucrose F68 7.2 KPO4 buffer AVS11 10% 20% 0.2% Pluronic1% arginine, 50 mM sucrose; F68 7.2 KPO4 buffer 10% raffinose AVS12 2020% 0.2% Pluronic 1% arginine, 50 mM raffinose F68 7.2 KPO4 buffer AVS1325 10% 0.2% Pluronic 5% arginine, 50 mM sucrose; F68 7.2 KPO4 buffer  2%raffinose AVS14 20 10% 0.2% Pluronic 10 mM methionine, sucrose F68 50 mM7.2 KPO4 buffer AVS15 20 10% 1% Gelatin 0.2% Pluronic 10 mM methioninesucrose F68 AVS16 20 10% 1% Gelatin 0.2% Pluronic 10 mM methioninesucrose F68 AVS17 20  2% 1% Gelatin 0.2% Pluronic 10 mM methionine,raffinose F68 AVS18 20 10% 0.2% Pluronic 10 mM methionine raffinose F68AVS19 20 10% 0.2% Pluronic 10 mM methionine, sucrose F68 100 mM 7.0citrate buffer AVS20 20 10% 1% Gelatin 0.2% Pluronic 10 mM methionine,sucrose F68 100 mM 7.0 citrate buffer AVS21 20 10% 1% Gelatin 0.2%Pluronic 10 mM methionine, sucrose, F68 100 mM 7.0 citrate  2% bufferraffinose AVS22 20  2% 1% Gelatin 0.2% Pluronic 10 mM methionine,raffinose F68 100 mM 7.0 citrate buffer AVS23 20 10% 0.2% Pluronic 10 mMmethionine, raffinose F68 100 mM 7.0 citrate buffer AVS24 20 10% 5%Gelatin 0.1% Pluronic 10 mM methionine, sucrose, F68 25 mM 7.2 KPO4  5%buffer raffinose AVS25 20 15% 5% Gelatin 0.1% Pluronic 10 mM methionine,sucrose F68 25 mM 7.2 KPO4 buffer AVS26 20 15% 5% Gelatin 0.1% Pluronic10 mM methionine, raffinose F68 25 mM 7.2 KPO4 buffer AVS27 20 10% 0.1%Pluronic 10 mM methionine, sucrose, F68 25 mM 7.2 KPO4  5% bufferraffinose AVS28 20 10% 5% Gelatin 0.1% Pluronic 2% ovalbumin, 10 mMsucrose, F68 methionine, 25 mM  5% 7.2 KPO4 buffer raffinose AVS29 40%5% Gelatin 0.1% Pluronic 10 mM methionine, raffinose F68 25 mM 7.2 KPO4buffer AVS30 10 40% 5% Gelatin 0.1% Pluronic 10 mM methionine, sucroseF68 25 mM 7.2 KPO4 buffer AVS31 10 40% 5% Gelatin 0.1% Pluronic 10 mMmethionine, sucrose F68 25 mM 7.2 KPO4 buffer, 1% glycerol AVS32 20 15%5% Gelatin 0.1% Pluronic 10 mM methionine, sucrose F68 25 mM 7.2 KPO4buffer AVS33 20 15% 1% Gelatin 0.1% Pluronic 10 mM methionine, sucroseF68 25 mM 7.2 KPO4 buffer AVS34 20 15% 0.1% Pluronic 10 mM methionine,sucrose F68 25 mM 7.2 KPO4 buffer AVS35 20 20% 5% Gelatin 0.1% Pluronic10 mM methionine, sucrose F68 25 mM 7.2 KPO4 buffer AVS36 20 20% 0.1%Pluronic 2% ovalbumin, 10 mM sucrose F68 methionine, 25 mM 7.2 KPO4buffer AVS37 10 20% 0.1% Pluronic 1% arginine, 10 mM sucrose, F68methionine, 25 mM 10% 7.2 KPO4 buffer raffinose AVS38 20 20% 1% Gelatin0.2% Pluronic 10 mM methionine, sucrose F68 25 mM 7.2 KPO4 buffer AVS3920 20% 5% Gelatin 0.005% 10 mM methionine, sucrose Tween 20 25 mM 7.2KPO4 buffer AVS40 20 20% 0.005% 2% ovalbumin, 10 mM sucrose Tween 20methionine, 25 mM 7.2 KPO4 buffer AVS41 10 40% 5% Gelatin 0.02% 10 mMmethionine, sucrose Pluronic F68 25 mM 7.2 KPO4 buffer AVS42 10 40% 5%Gelatin 0.02% 10 mM methionine, sucrose Pluronic F68 25 mM 7.2 KPO4buffer, 1% glycerol AVS43 40% 5% Gelatin 0.02% 10 mM methionine, sucrosePluronic F68 25 mM 7.2 KPO4 buffer AVS44 40% 5% Gelatin 0.02% 10 mMmethionine, sucrose Pluronic F68 25 mM 7.2 KPO4 buffer, 1% glycerolAVS45 10 40% 5% Gelatin 10 mM methionine,, sucrose 1% glycerol AVS46 50%1% Gelatin 10 mM methionine, sucrose 25 mM 7.2 KPO4 buffer, 1% glycerolAVS47 10 40% 5% Gelatin 0.02% 10 mM methionine, sucrose Pluronic F68 25mM 7.2 KPO4 buffer, 1% glycerol AVS48 40% 5% Gelatin 0.02% 10 mMmethionine, sucrose, Pluronic F68 25 mM 7.2 KPO4  5% buffer, 1% glyceroltrehalose AVS49 40% 3% Gelatin 0.02% 10 mM methionine, sucrose PluronicF68 25 mM 7.2 KPO4 buffer AVS50 40% 1% Gelatin 0.02% 10 mM methionine,sucrose Pluronic F68 25 mM 7.2 KPO4 buffer AVS51 40% 0.02% 10 mMmethionine, sucrose Pluronic F68 25 mM 7.2 KPO4 buffer AVS52 40% 5%Gelatin 10 mM methionine, sucrose 25 mM 7.2 KPO4 buffer AVS53 40% 5%Gelatin 0.02% 25 mM 7.2 KPO4 sucrose Pluronic F68 buffer AVS54 40% 5%Gelatin 25 mM 7.2 KPO4 sucrose buffer AVS55 20% 5% Gelatin 0.02% 10 mMmethionine, sucrose Pluronic F68 25 mM 7.2 KPO4 buffer AVS56 10% 5%Gelatin 0.02% 10 mM methionine, sucrose Pluronic F68 25 mM 7.2 KPO4buffer AVS57 40% 0.02% 5% ovalbumin, 25 mM sucrose Pluronic F68 7.2 KPO4buffer AVS58A 40% 5% Gelatin 0.02% 25 mM 7.2 KPO4 sucrose (Sigma AD)Pluronic F68 buffer AVS58B 40% 5% Gelatin 0.02% 25 mM 7.2 KPO4 sucroseSigma (R) Pluronic F68 buffer AVS59 40% 5% Gelatin 0.02% 10 mMmethionine, sucrose Pluronic F68 25 mM 7.2 KPO4 buffer AVS60 40% 5%Gelatin 0.02% 10 mM methionine, sucrose Pluronic F68 25 mM 7.2 KPO4buffer AVS61 20% 2.5% Gelatin 0.01% 5 mM methionine, sucrose PluronicF68 12.5 mM 7.2 KPO4 buffer AVS62 40% 0.02% 5% arginine, 10 mM sucrosePluronic F68 methionine, 25 mM 7.2 KPO4 buffer AVS63 40% 2.5% PEG 10 mMmethionine, sucrose 1000, 0.02% 25 mM 7.2 KPO4 Pluronic F68 buffer AVS6440% 2.5% PVP 10 mM methionine, sucrose 10,000, 0.02% 25 mM 7.2 KPO4Pluronic F68 buffer AVS65 40% 2.5% Ficoll 10 mM methionine, sucrose400K, 0.02% 25 mM 7.2 KPO4 Pluronic F68 buffer AVS66 20% 2% Gelatin0.02% 10 mM methionine, sucrose Pluronic F68 25 mM 7.2 KPO4 buffer AVS6740% 5% Gelatin 0.02% 1% methionine, 25 mM sucrose Pluronic F68 7.2 KPO4buffer AVS68 20% 2% Gelatin 0.02% 1% methionine, 25 mM sucrose PluronicF68 7.2 KPO4 buffer AVS69 40% 0.02% 5% arginine, 1% sucrose Pluronic F68methionine, 25 mM 7.2 KPO4 buffer AVS70 20% 0.02% 5% arginine, 1%sucrose Pluronic F68 methionine, 25 mM 7.2 KPO4 buffer

The thermostability of the above formulations after post-lyophilizationstorage at 37° C. or 50° C. were measured. Increased thermostability canbe observed as shown as a decrease in the rate of potency loss, measuredas the log FFU/ml or TCID₅₀/ml. The time required for a one log orderloss in FFU/ml are provided below for various formulations of theinvention:

Formulation Stability at 37° C. AVS43 125 days AVS41  97 days AVS44 142days AVS30  93 days AVS31  48 days AVS42 146 days

Example 3 Foam Drying Conditions

Formulations were prepared using the following lyophilization/dryingchamber conditions:

Cycle 1: 1) Pre-cool shelves to 25° C.; 2) Load vials and allow toequilibrate; 3) Set vacuum to 50 mTorr; 4) Hold for 30 minutes; 5) Rampto 45° C.; 6) Hold for 1 hour; 7) Adjust the temperature to 37° C. andhold for 1 hour; and 8) Stopper vials. Cycle 2: 1) Pre-cool shelves to30° C.; 2) Load vials and allow to equilibrate; 3) Set vacuum to 50mTorr; 4) Hold for 2 hours; 5) Ramp to 37° C.; 6) Hold for 16 hours; and7) Stopper vials. Cycle 3: 1) Pre-cool shelves to 15° C.; 2) Load vialsand allow to equilibrate; 3) Set vacuum to 50 mTorr; 4) Hold for 60minutes; 5) Ramp to 37° C.; 6) Hold for 20 hours; and 7) Stopper vials.Cycle 4: 1) Pre-cool shelves to 12° C.; 2) Load vials and allow toequilibrate; 3) Set vacuum to 50 mTorr; 4) Hold for 25 minutes; 5) Rampto 33° C.; 6) Hold for 24 hours; and 7) Stopper vials. Cycle 5: 1)Pre-cool shelves to 17° C.; 2) Load vials and allow to equilibrate for10 minutes; 3) Set vacuum to 50 mTorr; 4) Hold for 60 minutes; 5) Rampto 37° C.; 6) Hold for 48 hours; 7) Ramp to 40° C.; 8) Hold for 48hours; and 9) Stopper vials. Cycle 6: 1) Pre-cool shelves to 20° C.; 2)Load vials and allow to equilibrate; 3) Set vacuum to 50 mTorr; 4) Holdfor 60 minutes; 5) Ramp to 33° C.; 6) Hold for 72 hours; and 7) Stoppervials.

Example 4 Formulations

The drying cycle shown in Example 1 was employed to stabilize liveB/Harbin influenza virus. The following table summarizes the observedstability profiles of these formulations after storage for ten months at25° C.:

Months Process Pluronic to 1 log Loss KPO4 buff. Sucrose GelatinMethionine F68 Slope FFU/ml (Log Formulation pH 7.2 (mM) (%) (%) (mM)(%) at 25° C. loss FFU/ml) AVS068 25 10 0 0 0.02 −0.167 6 0.20 AVS071 2510 0 10 0 −0.245 4.1 0.13 AVS072 25 10 0 66.7 0.2 −0.047 21.4 0.37AVS073 25 10 2 0 0.2 −0.089 11.2 0.63 AVS074 25 10 2 10 0.02 −0.056 17.81.13 AVS075 25 10 2 66.7 0 −0.154 6.5 0.43 AVS076 25 10 5 0 0 −0.145 6.90.67 AVS077 25 10 5 10 0.2 −0.008 121.2 0.47 AVS078 25 10 5 66.7 0.02−0.050 20.0 0.27 AVS079 25 10 5 66.7 0.2 −0.043 23.1 0.83 AVS080 25 20 00 0 −0.132 7.6 0.50 AVS081 25 20 0 10 0.02 −0.060 16.8 0.53 AVS082 25 200 66.7 0.2 −0.045 22.4 0.07 AVS083 25 20 2 0 0.02 −0.040 24.9 2.27AVS084 25 20 2 10 0 −0.096 10.4 0.33 AVS085 25 20 2 66.7 0.2 −0.040 25.00.43 AVS086 25 20 5 0 0.2 −0.022 44.6 0.30 AVS087 25 20 5 10 0.2 −0.005200.4 0.40 AVS088 25 20 5 66.7 0 −0.060 16.7 −0.07 AVS089 25 20 5 66.70.02 −0.022 44.5 0.67 AVS090 25 40 0 0 0.2 −1.283 0.8 0.63 AVS091 25 400 10 0.2 −1.117 0.9 0.77 AVS092 25 40 0 66.7 0 −0.120 8.3 0.50 AVS093 2540 0 66.7 0.02 −0.033 30.2 0.73 AVS094 25 40 2 0 0 −0.116 8.6 0.83AVS095 25 40 2 10 0.2 −0.016 63.8 0.47 AVS096 25 40 2 66.7 0.02 −0.03033.2 0.80 AVS097 25 40 2 66.7 0.2 −0.017 58.1 0.27 AVS053s 25 40 5 00.02 −0.022 44.6 0.47 AVS098 25 40 5 0 0.2 −0.023 43.4 0.37 AVS052b 2540 5 10 0 −0.087 11.6 0.67 AVS043c 25 40 5 10 0.02 −0.037 27.2 0.77AVS099 25 40 5 66.7 0 −0.067 15.0 0.40

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, the formulations, techniques and apparatusdescribed above can be used in various combinations. All publications,patents, patent applications, and/or other documents cited in thisapplication are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication, patent,patent application, and/or other document were individually indicated tobe incorporated by reference for all purposes.

1. A method for preparing a dry foam composition comprising a bioactivematerial, which method comprises: preparing a formulation comprising ina solvent: the bioactive material, a foaming agent, and a polyol orpolymer; expanding the formulation into a foam; and, stabilizing ordrying the foam by evaporating or sublimating the solvent from the foam;thereby preparing a dry foam composition.
 2. The method of claim 1,wherein the bioactive material is selected from the group consisting ofpeptides, proteins, nucleic acids, antibodies, vaccines, bacteria,viruses, liposomes, platelets, and cell suspensions.
 3. The method ofclaim 2, wherein the viruses are live viruses selected from the groupconsisting of influenza virus, parainfluenza virus, AAV, adenovirus,respiratory syncytial virus, herpes simplex virus, cytomegalovirus, SARSvirus, corona virus family members, human metapneumovirus, andEpstein-Barr virus.
 4. The method of claim 1, wherein the foaming agentcomprises a gas in solution in the formulation, a high vapor pressuresolvent, a carbonate, an active metal, or a suspension of gas bubbles.5. The method of claim 1, wherein the polyol is present in an amountranging from about 20 weight percent to about 45 weight percent.
 6. Themethod of claim 1, wherein the polyol comprises sucrose, trehalose,sorbose, melezitose, sorbitol, stachyose, raffinose, fructose, mannose,maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose,glucose, mannitol, xylitol, erythritol, threitol, sorbitol, glycerol, orL-gluconate.
 7. The method of claim 1, wherein the polymer is present inan amount ranging from about 1 weight percent to about 10 weightpercent.
 8. The method of claim 1, wherein the polymer compriseshydrolyzed gelatin, unhydrolyzed gelatin, collagen, chondroitin sulfate,water soluble polymers, polyvinyl pyrrolidone, a sialatedpolysaccharide, actin, myosin, microtubules, dynein, kinetin, or humanserum albumin.
 9. The method of claim 1, wherein expanding into a foamcomprises, degassing the formulation, boiling the formulation, forming agas by chemical reaction, forming bubbles by electrolysis, expandingbubbles suspended in the formulation, or injection of bubbles into theformulation.
 10. The method of claim 1, wherein the formulation furthercomprises a surfactant or a buffer.
 11. The method of claim 1, whereinthe bioactive material comprises a lipid membrane, and wherein themethod further comprises cooling the formulation to a phase transitiontemperature of the lipid membrane.
 12. The method of claim 11, furthercomprising holding the formulation at the phase transition temperaturefor from about 10 minutes to about 60 minutes.
 13. The method of claim1, further comprising increasing the temperature of the formulation to adrying temperature that is less than or about the glass transitiontemperature of the dry foam.
 14. The method of claim 1, furthercomprising grinding the dry foam to a powder comprising an averageparticle size from about 0.1 um to about 100 um.
 15. The method of claim14, wherein the a powder comprises an average particle size from about50 um to about 100 um.
 16. The method of claim 1, further comprisingadministering the dry foam composition to a mammal as a reconstitutedliquid or as a ground powder.
 17. The method of claim 16, whereinadministering comprises delivering the composition to the mammal byintravenous, intramuscular, intraperitoneal, intracerebrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, intranasal, or pulmonary routes.
 18. The method of claim 1,further comprising secondary drying the foam at a temperature from about10° C. to about 70° C.
 19. The method of claim 1, wherein said preparingthe formulation comprises providing sufficient polyol so that a glass isformed upon said drying step.
 20. The method of claim 1, wherein theexpanding is other than by boiling.
 21. The method of claim 1, whereinthe foaming agent is other than the solvent.
 22. The method of claim 1,wherein said expansion is by action of the foaming agent.